The hypothesis is that gastric acid secretion may involve two separate steps, viz. formation of "acid" (protons, buffered protons or an enzyme) that is stored in an intracellular compartment and translocation (i.e., secretion) of protons from this storage pool by exocytosis to the external medium. Secretagogues (histamine, forskolin and cAMP), inhibitors (H2-antagonists and omeprazole) affect the formation of "acid" and cause changes in the total amount of acid secreted (non-conservative effect), while nitrite and low concentration of thiocyanate affect the translocation step and thus, change the shape of the acid secretion curve but not the total amount of acid secreted conservative effect. The specific goal of this proposal is to determine the nature and location of acid storage compartments (e.g., vesicular proton storage, H+pumps), and the mechanisms which regulate the storage and release of acid by the use of specific secretagogues and inhibitors. To this end, the dynamics of acid formation and secretion, and the associated cellular events will be studied. Methodology: The acid secretion rate in vitro frog gastric mucosa, mounted in modified Ussing chambers, will be continuously monitored and recorded by a pH-stat interfaced with a computer, and anlayzed for net amount of acid secreted, and for time of onset of stimulation/inhibition. Acridine orange, a fluorescent weak base, will be used to detect changes in intra- cellular acid compartments in response to stimulators/inhibitors. FITC-Dextran (a fluorescent probe) will be used for characterization of endocytotoic and exocytotic processes. Both probes will be studied in perfused preparations while simulataneoulsy both acid secretion and the fluorescence of secretory effluent will be monitored in a flowthrough system. Both probes will also be studied in perfused tissues using fluorescence microsocpy, the fluorescence recorded photographically or with a computer-assisted fluorometer. Correlation of the movement of these probes with the acid secretory pattern will be studied to establish the relationship between acid secretion and exocytosis. Immunocytochemical electron microscopy will be used to visualize acidic intracellular compartments using DAMP, monoclonal antibodies directed towards DAMP and gold conjugated IgG as the second antibody.